PROJECT SUMMARY/ABSTRACT Sprouting angiogenesis, the formation of blood vessel sprouts, is essential for building the complex and highly branched vascular tree required to sustain life. This process also plays pivotal roles in wound healing and organ regeneration. In addition, too much, too little or otherwise abnormal sprout formation is associated with many diseases. Thus, there is much interest in the therapeutic manipulation of sprouting angiogenesis. However, to achieve this we must first dissect the molecular mechanisms that regulate this vital process. This is precisely the overarching goal of this pioneering proposal, the first to address how the Vascular Endothelial Growth Factor (VEGF) and Semaphorin-PlexinD1 (Sema-PlxnD1) pathways interact via three shared molecular components and determine fundamental aspects of sprout formation, such as the abundance, positioning and shape of vascular branches. The experiments described here exploit the complementary advantages of the zebrafish and cultured endothelial cells as model systems for sprouting angiogenesis, employ innovative fluorescent reporters of post-transcriptional gene expression and novel zebrafish mutants of each of the three shared pathway components. By elucidating how VEGF and Sema-PlxnD1 interact the present study will provide key insights into the molecular mechanisms that endothelial cells use to integrate opposing inputs during sprout formation and suggest novel molecular targets for therapeutic manipulation of this process. Such new therapies could help to preserve the sight of people at risk for blindness due to excessive corneal vascularization (exudative macular degeneration), restore proper blood supply to those suffering from ischemia and limit the growth and spread of tumors in many cancer patients by normalizing their tumor vasculature.